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Thursday, May 5, 2011

Surface Plasmon Resonance and its Biosensor / Nanotechnology Applications

Surface plasmon resonance (SPR) is a phenomenon occurring at metal surfaces(typically gold and silver) when an incident light beam strikes the surface at a particular angle.Depending on the thickness of a molecular layer at the metal surface,the SPR phenomenon results in a graded reduction in intensity of the reflected light.Biomedical applications take advantage of the exquisite sensitivity of SPR to the refractive index of the medium next to the metal surface, which makes it possible to measure accurately the adsorption of molecules on the metal surface or on to surface of metal nanoparticles and their eventual interactions with specific ligands. It is the fundamentals behind many color based biosensor applications and different lab-on-a-chip sensors.


The underlying physical principles of SPR are complex.Fortunately, an adequate working knowledge of the technique does not require a detailed theoretical understanding. It suffices to know that SPR-based instruments use an optical method to measure the refractive index near (within ~300 nm) a sensor surface. In the BIAcore this surface forms the floor of a small flow cell, 20-60 nL in volume , through which an aqueous solution (henceforth called the running buffer) passes under continuous flow (1-100 ┬ÁL.min-1). In order to detect an interaction one molecule (the ligand) is immobilised onto the sensor surface. Its binding partner (the analyte) is injected in aqueous solution (sample buffer) through the flow cell, also under continuous flow. As the analyte binds to the ligand the accumulation of protein on the surface results in an increase in the refractive index. This change in refractive index is measured in real time, and the result plotted as response or resonance units (RUs) versus time (a sensorgram). Importantly, a response  (background response) will also be generated if there is a difference in the refractive indices of the running and sample buffers. This background response must be subtracted from the sensorgram to obtain the actual binding response. The background response is recorded by injecting the analyte through a control or reference flow cell, which has no ligand or an irrelevant ligand immobilized to the sensor surface. One RU represents the binding of approximately 1 pg protein/mm2. In practise >50 pg/mm2 of analyte binding is needed. Because is it very difficult to immobilise a sufficiently high density of ligand onto a surface to achieve this level of analyte binding, BIAcore have developed sensor   - 4 - surfaces with a 100-200 nm thick carboxymethylated dextran matrix attached. By effectively adding a third dimension to the surface, much higher levels of ligand immobilisation are possible. However, having very high levels of ligand has two important drawbacks. Firstly, with such a high ligand density the rate at which the surface binds the analyte may exceed the rate at which the analyte can be delivered to the surface (the latter is referred to as mass transport). In this situation, mass transport becomes the rate-limiting step. Consequently, the measured association rate constant (kon) is slower than the true kon. A second, related problem is that, following dissociation of the analyte, it can rebind to the unoccupied ligand before diffusing out of the matrix and being washed from the flow cell. Consequently, the measured dissociation rate constant (apparent koff) is slower than the true koff. Although the dextran matrix may exaggerate these kinetic artefacts (mass transport limitations and re-binding) they can affect all surface-binding techniques . 

Surface Plasmon Resonance stems one of the basic principles of optics, that of total internal reflectance (or TIR).
  • Occurs when a thin conducting film is placed at the interface between the two optical media.
  • At a specific incident angle, greater than the TIR angle, the surface plasmons in the conducting film resonantly couple with the light because their frequencies match.
SPR is good for:
  • Evaluation of macromolecules.
  • Equilibrium measurements (affinity and enthalpy).
  • Kinetic measurements. 
  • Analysis of mutant proteins.
SPR is not good for:

  • High throughput assays.
  • Concentration assays
  • Studying small analytes.
Some of the potential areas of application include
  • Medical diagnostics
  • Environmental monitoring
  • Agriculture pesticide and antibiotic monitoring
  • Food additive testing
  • Military and civilian airborne
  • Biological and chemical agent testing
  • Real time chemical and biological production process monitoring.

Protocol for working in BIAcore can be obtained from the below link